Method for preparing peptides and intermediate products

ABSTRACT

Peptides having the carboxyl terminated sequence of the  pancreozymin-cholstokinin 25-33 modified such that the amino acid group 25 carries a strongly basic group, the amino acid group 28 exhibits a hydrophilic character similar to that of the hydroxyamino acids and the amino acid group 31 exhibits a strongly hydrophobic character similar to that of amino acids with aliphatic side chains, and in particular nonapeptides having the sequence H--X--Asp--Tyr(SO 3  H)--Y--Gly--Trp--Z--Asp--Phe--NH 2  wherein X is arginine, homoarginine, norarginine, N.sub.ε,N.sub.ε -dialkyllysine, N.sub.δ  or N.sub.δ -dialkyl-ornithine, Y is threonine, serine or hydroxy-proline and Z is norleucine, leucine, norvaline or α-amino-butyric acid, possess pronounced pancreozymin activity and can be employed in pharmaceutical preparations for controlling the function of the gall bladder and for controlling the enzyme secretion of the pancreas. Tyrosine-O-sulfate-barium salt and its N-acyl derivatives can be used as intermediate products for the preparation of peptides. For introducing the amino acid group 27 an N-acyltyrosine is reacted with an excess of pyridine--SO 3  in a polar organic solvent, the resulting solution is extracted with water, the barium salt of the N-acyl-tyrosine-O-sulfate is precipitated from the aqueous phase by addition of a soluble barium compound, possibly the acyl group is split off in conventional manner, and the resulting tyrosine-O-sulfate-barium salt or its acyl derivative are processed employing the usual methods of peptide synthesis.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.331,311, filed Dec. 16, 1981, now abandoned, which in turn is adivisional application of application Ser. No. 145,500, filed Apr. 29,1980, now U.S. Pat. No. 4,368,192, granted Jan. 11, 1983.

DESCRIPTION

1. Field of the Invention

The present invention relates to the preparation of peptides containinga tyrosine-O-sulfate moiety.

2. Brief Description of the Background of the Invention Including PriorArt

Tyrosine-O-sulfate was discovered as a metabolite in the urine ofmammals and was later also discovered as a building block in variousbiologically active peptides and proteins, for example in gastrines.This amino acid group further provides the "essential" grouping forbiological activity in the biologically interesting peptide hormonescholecystokinin-pancreozymin and caerulein. Therefore, it is animportant goal of biochemical and in particular peptide-chemicalresearch and of the pharmaceutical development to synthesize suchbiologically active tyrosine-O-sulfate containing peptides and proteins,respectively.

Biologically active peptides containing tyrosine-O-sulfate have beenprepared predominantly by subsequent sulfation of the phenolic groupwithin the peptide with concentrated sulfuric acid or pyridine/SO₃complex. These methods have the disadvantage that to an interferingextent side reactions occur such as sulfonation of the phenolic ring, ofthe indolyl or imidazolyl groups on the one hand or reactions with themethionine-thioether-grouping, the arginine-guanidine and the primaryamido-function on the other hand (J. Am. Chem. Soc. 68,1024 and 1031(1946)). The additional easily sulfatable hydroxy and amino functionshad to be protected. This disadvantage can be avoided if it isachievable to construct such active peptides with tyrosine-O-sulfate asa starting material. This would allow to avoid the limitations on thepeptide synthesis imposed by the cited side reactions. An experiment hasbeen reported to build such peptides employing tyrosine-O-sulfate as thepotassium sulfate, however neither experimental details nor the resultsare known (Experientia 28, 7 (1972)).

SUMMARY OF THE INVENTION

1. Purposes of the Invention

It is an object of the invention to provide intermediates and a methodfor synthesizing peptides containing a tyrosine-O-sulfate group.

2. Brief Description of the Invention

The present invention provides tyrosine-O-sulfate-barium salt and itsacyl derivatives which can be prepared by contacting N-acyl-tyrosinewith excess pyridine-SO₃ in a polar organic solvent or solvent mixture,extracting the solution with water, adding to the resulting aqueousphase a soluble barium compound and separating from the aqueous phasethe precipitated barium salt of the N-acyl-tyrosine-O-sulfate. Thepresent invention will be illustrated by a method for preparingpancreozymincholecystokinin active peptides having a modified carboxylterminated sequence of pancreozymin-cholecystokinin 25-33 wherein themodification comprises the amino acid group 25, which carries a stronglybasic group, the amino acid group 28, which exhibits a hydrophiliccharacter similar to that of hydroxyamino acid groups, and the aminoacid group 31, which exhibits a strongly hydrophobic character similarto that of amino acid groups having an aliphatic side chain.N-acyl-tyrosine is reacted in a polar solvent with an excess ofpyridine-SO₃, the resulting solution is extracted with water, the bariumsalt of the N-acyl-tyrosine-O-sulfate is precipitated from the aqueousphase by adding a soluble barium compound and the resultingN-acyl-tyrosine-O-sulfate-barium salt is processed for obtaining thedesired compounds by conventional peptide synthesis methods. The acylgroup can be split off from the N-acyl-tyrosine-O-sulfate-barium saltand form tyrosine-O-sulfate-barium salt. The tyrosine-O-sulfate-bariumsalt can be processed by condensation in accordance with thedicyclohexylcarbodiimide/1-hydroxybenzotriazole method, in accordancewith the method via mixed anhydrides or in accordance with the methodemploying dicylohexylcarbodiimide/N-hydroxysuccinimide.

A nonapeptide can be prepared by condensing tyrosine-O-sulfate-bariumsalt with H--Y(t--Bu)--Gly--Trp--Z--Asp(OtBu)--Phe--NH₂,hydrogenolytically splitting off the benzyloxycarbonyl group from thecondensation product; extending the resulting tyrosine-O-sulfate-peptidederivative with B--X(B₂)--Asp(OtBu)--OH in accordance with thedicyclohexylcarbodiimide/N-hydroxysuccinimide method to obtainB--X(B₂)--Asp(OtBu)--Tyr(SO₃Ba_(1/2))--Y(tBu)--Gly--Trp--Z--Asp(OtBu)--Phe--NH₂, wherein X isarginine, homoarginine, N.sub.ε, N.sub.ε -dialkyllysine, N.sub.δ orN.sub.δ -dialkyl-ornithine, Y is threonine, serine or hydroxy-proline,and Z is norleucine, norvaline, or α-amino-butyric acid. The threebenzyloxycarbonyl groups (B) are hydrogenolytically split off from theamino terminated X-group and the tertiary butanol protective groups aresplit off from the resulting nonapeptide with an acid reagent,preferably trifluoro-acetic acid. A preferred nonapeptide has thesequence H--Arg--Asp--Tyr(SO₃ H)--Thr--Gly--Trp--Nle--Asp--Phe--NH₂.

DESCRIPTION OF INVENTION AND PREFERRED EMBODIMENTS

A nonapeptide which may be prepared in accordance with the presentinvention may have the general formula H--X--Asp--Tyr(SO₃H)--Y--Gly--Trp--Z--Asp--Phe--NH₂, wherein X is arginine, homoarginine,norarginine, N.sub.ε,N.sub.ε -dialkyllysine, N.sub.δ or N.sub.δ-dialkyl-ornithine, Y is threonine, serine or hydroxy-proline and Z isnorleucine, norvaline or α-amino-butyric acid, or less preferablyleucine.

A preferred sequence is H--Arg--Asp--Tyr (SO₃H)--Thr--Gly--Trp--Nle--Asp--Phe--NH₂. The peptides are useful aspancreozymin-cholecystokinin active compositions.

The preparation of the peptides according to the present inventionemploys conventional steps as they are, for example, disclosed inMoroder, L., L. Wilschowitz, E. Jaeger, S. Knof, P. Thamm and E. Wuenschin "Hormonal Receptors in Digestive Tract Physiology" (G. Rosselin etal. eds.), Elsevier/North-Holland Biomedical Press, Amsterdam 1979,Pages 129-135 and also Moroder, L., L. Wilschowitz, E. Jaeger, S. Knof,P. Thamm and E. Wuensch (1979) Hoppe Seyler's Z. Physiol. Chem. 360,787-790.

A preferred method for making the peptides comprises reacting anN-acyl-tyrosine with excess pyridine-SO₃ in a polar organic solvent forintroducing the amino acid group 27, extracting the resulting solutionwith water, adding a soluble barium compound to the aqueous phase andprecipitating the barium salt of the N-acyl-tyrosine-O-sulfate, ifdesired splitting off the acyl group in a conventional way andprocessing the resulting tyrosine-O-sulfate-barium salt or its acylderivative by generally known steps of the usual peptide synthesismethods.

Tyrosine-O-sulfate-barium salt and/or an acyl derivative thereof andpreferably N-carbobenzoxy-tyrosine-O-sulfatebarium salt is employed asan intermediate in accordance with the present invention.

It is possible to avoid the disadvantages of the prior art by employingthe intermediate product of the present invention, thetyrosine-O-sulfate-barium salt and its N-acyl derivatives together withthe acyl protective groups usually employed in peptide chemistry andpreferably of the N-carbobenzoxy-tyrosine-O-sulfate-barium salt. Thustyrosine-O-sulfate containing compounds are provided which are useful asstarting materials for the synthetic production of peptides. Based ontheir solubility characteristics they facilitate the isolation of thepeptides prepared with them.

Tyrosine-O-sulfate-barium salt has the formula H--Tyr(SO₃ Ba_(1/2))--OH,the cited carbobenzoxy compound the formula B--Tyr(SO₃Ba_(1/2))--O--Ba_(1/2). In these formulas Tyr means tyrosine and Brepresents the carbobenzoxy group. The abbreviations employed hereincorrespond to the rules set forth in Houben-Weyl, Methoden derorganischen Chemie, 4th edition, Volume 15/1, page 20, publisher Thieme,Stuttgart (1974). Houben-Weyl cites also additional acyl protectivegroups usual in peptide chemistry. Examples are N-t-butyloxycarbonyl-,N-fluorenyloxycarbonyl- andN-2-nitrophenylsulfenyl-tyrosine-O-sulfate-barium salt.

Tyrosine-O-sulfate-barium salt is prepared in accordance with thepresent invention by reacting an N-acyltyrosine with excess pyridine-SO₃in a polar organic solvent or solvent mixture, extracting the resultingsolution with water, precipitating the barium salt of theN-acyltyrosine-O-sulfate by addition of a soluble barium compound andsplitting off the acyl group, if desired. A preferred organic solvent ispyridine or a mixture of pyridine-dimethylformamide. However otherweakly basic or neutral polar organic solvents and solvent mixtures canalso be employed. The excess of pyridine-SO₃ complex is not critical, a2 to 6 fold amount can be employed and preferably a 3 to 5 fold amountcan be employed relative to the SO₃ equivalents. The reaction can beperformed at temperatures from about 0° C. to the boiling point of thesolvent and preferably at temperatures of from about 20° C. to 80° C.After completion of the reaction the excess pyridine-SO₃ complex isseparated by cooling and removed from the solution. Then the solution isthinned with water, impurities are extracted with organic solvents suchas acetic acid ethyl ester and then a suitable soluble barium compound,preferably barium hydroxide, is added to the aqueous phase. It ispreferred to employ a 2 to 3 fold excess of the barium compound.

Excess barium hydroxide can be separated by addition of carbon dioxide,while acidification of the solution is avoided by addition of weakalkaline materials which are preferably organic bases such as pyridine.After removal of the precipitate the acyl group containing barium saltof the tyrosine-O-sulfate is obtained. It can be employed as is for thesynthesis of peptides or it can be transformed into thetyrosine-O-sulfate-barium salt. The acyl group is split off in the wayusually employed in peptide synthesis, for example in case of acarbobenzoxy group by hydrogenation in the presence of palladium as acatalyst preferably in a reaction medium containing dimethylformamide.

The new barium salts of the present invention are especially suitablefor the total synthetic formation of tyrosine-O-sulfate containingpeptides and proteins, respectively, since they influence positively theisolation of the peptide derivatives prepared with them. It wassurprisingly discovered that the usual methods of peptide synthesis canbe employed without splitting off the O-sulfate group. This is valid forexample also for the splitting off of the protective groups such as thesplitting off of protective groups based on tertiary butanol under acidconditions. This was unexpected, since in the past reference was alwaysmade to the instability of tyrosine-O-sulfate against acids.

Typical examples of peptide synthesis methods performable in thepresence of the compounds of the present invention without decomposingthe same are the condensation in accordance with thedicyclohexylcarbodiimide/1-hydroxybenzotriazole method(DCCD/HOBT-method), the methods via mixed anhydrides for example withpivaloylchloride, chloroformic acid ethyl ester or isobutylester.Another example for a suitable method of synthesis is thedicyclohexylcarbodiimide/N-hydroxysuccinimide method.

The particular usefulness of the intermediate products of the presentinvention is demonstrated with the aid of the benzyloxycarbonyl groupcontaining barium salt in an example of synthesizing the nonapeptide ofthe sequence H--Arg--Asp--Tyr(SO₃ H)--Thr--Gly--Trp--Leu--Asp--Phe--NH₂.According to the DCCD/HOBT method the barium compound of the presentinvention was condensed withH--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂ and gave clearlyB--Tyr(SO₃ Ba_(1/2))--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂ (III)with a yield of 70%. The hydrogenolytically splitting off of thebenzyloxycarbonyl group from (III) under usual hydrogenation conditionsproduced H--Tyr(SO₃Ba_(1/2))--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂ (IV) with ayield of 96%. A successive extension of the "amino free"tyrosine-O-sulfate peptide derivative (IV) withB--Arg(B₂)--Asp(OtBu)--OH in accordance with the DCCD/HOSU method forobtaining B--Arg(B₂)--Asp(OtBu)--Tyr(SO₂Ba_(1/2))--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH. (V) had a yieldof 96%. The hydrogenolytically splitting off of the threebenzyloxycarbonyl groups from the amino terminated arginine group at apH of about 6 provided the desired nonapeptide derivative (VI), i.e.H--Arg--Asp(OtBu)--Tyr(SO₃H)--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂ with 83% yield.

Despite the numerous references in the prior art to the acid instabilityof tyrosine-O-sulfate and its peptides also the last step was successfulin the preparation of the pancreozymin-cholecystokinin analog, i.e., thesplitting off of the protective groups based on tertiary butanol. Thefeared simultaneous partial splitting off of the sulfate-semi-estergrouping was not observed. For the splitting off of the protectivegroups the acid means known for this purpose, are suitable, andtrifluoroacetic acid of from about 70 to 90 percent concentration ispreferred. It is preferred to add a cation captor such as, for example,2-methylindol.

EXAMPLE 1 B-Tyr(SO₃ Ba_(1/2))--O--Ba_(1/2).3H₂ O (I)

12.7 g (40.3 mmole) B--Tyr--OH in pyridine are contacted with 25.8 g(161.1 mmole) pyridine-SO₃ complex. The suspension is heated to 60° C.and agitated at this temperature until the complex is dissolved (about1/2 hour). The solution is then cooled to 0° C. and filtered, thefiltrate is concentrated in vacuum and filtered again in order toseparate the excess of the complex. Then the solution is thinned withwater and extracted twice with acetic acid ethyl ester and the separatedaqueous phase is saturated with nitrogen and dependent on the amount ofthe complex previously separated from about 2 to 3 equivalents of bariumhydroxide are added.

The precipitate is sucked off and the excess of barium hydroxide isremoved by introduction of carbon dioxide. Addition of pyridine preventsa lowering of the pH to below 7.

After filtration the solution is concentrated to about 100 ml and theproduct is precipitated with ethanol. Chromatographically pure inn-BuOH/AcOH/H₂ O/acetic acid ethyl ester (3:1:1:5).

[α]₅₄₆ ²⁰ : +22.7° and [α]_(D) ²⁰ : +18.9° (c=1, in DMF).

Yield: 21.34 g (91 percent of theoretical).

C₁₇ H₁₅ NO₈ SBa.3H₂ O (584.80): Calculated: C: 34.92, H: 3.62, N: 2.39,Ba: 25.88, Found: C: 34.87, H: 3.10, N: 2.20, Ba: 23.20.

Calculated residue: 39.9 percent (as BaSO₄). Found ignition residue 40.5percent (as BaSO₄).

EXAMPLE 2 H--Tyr(SO₃ Ba_(1/2))--OH.H₂ O (II)

5 g (8.55 mmole) B--Tyr(SO₃ Ba_(1/2))--O--Ba_(1/2).3H₂ O are as usualhydrogenated in the presence of a palladium catalyst indimethylformamide. After completion of the reaction the filtrate isconcentrated in vacuum and the residue is dissolved in water. Theinsoluble materials are filtered off and the product is precipitatedwith ethanol. The product is dissolved in water and the pH is brought to6.5 by introducing carbon dioxide. The precipitate (BaCO₃) is removed byfiltration and the filtrate is concentrated and finally the product isprecipitated with ethanol. Chromatographically pure in n-BuOH/AcOH/H₂O/acetic acid ethyl ester (3:1:1:5): melting point 232° C.

[α]_(D) ²⁰ : -24.8° and [α]₅₄₆ ²⁰ : -29.6° (c=1, in DMF/H₂ O; 95:5 byvolume).

Yield: 2.6 g (84 percent of theoretical).

C₉ H₁₀ NO₆ SBa₀.5.H₂ O (346.94): Calculated: C: 31.15, H: 3.48, N: 4.04,Found: C: 30.99, M: 3.12, N: 3.96.

Residue: calculated: 32.18 percent, found: 33.64 percent.

EXAMPLE 3 A. B--Tyr(SO₃Ba_(1/2))--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂ (III)

0.62 ml (0.48 mmole) of a 0.77 n HCl solution in dioxane is added dropby drop to a solution of 0.28 g (0.48 mmole) B--Tyr(SO₃Ba_(1/2))--OBa_(1/2) 3H₂ O in dimethylformamide. Under agitation 0.35 g(0.40 mmole) H--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂.H₂ O, 70 mg(0.52 mmole) 1-hydroxybenzotriazole and finally, at -10° C., 99 mg (0.48mmole) of dicyclohexylcarbodiimide are added. After 6 hours at -4° C.and 6 hours at room temperature the solvent is removed by vacuum and theresidue is digested with ether. The raw product is taken up withdimethylformamide, the insoluble parts are filtered off and the productis precipitated with water. The product is again precipitated from amethanol solution with ether after the insoluble had been filtered off.

Chromatographically pure in n--BuOH/AcOH/H₂ O/acetic acid ethyl ester(3:1:1:5); melting point: 168° C. (decomposition).

[α]₅₄₆ ²⁰ : -22.7° (c=1, in dimethylformamide), [α]_(D) ²⁰ : -18.8°.

C₆₁ H₇₈ N₉ O₁₆ SBa₀.5 (1294.11): Calculated: C: 56.62, H: 6.08, N: 9.79,Found: C: 56.64, H: 6.21, N: 9.87.

B. H--Tyr(SO₃ Ba_(1/2))--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂/1H₂ O.1DMF

4.07 g (3.14 mmole) B--Tyr(SO₃Ba_(1/2))--Thr(tBu)--Gly--Trp--Leu--Asp--(OtBu)--Phe--NH₂ are as usualhydrogenated in the presence of palladium in dimethylformamide. Afterremoval of the catalyst the solvent is evaporated in vacuum and theresidue is triturated with ether.

Chromatographically pure in n--BuOH/AcOH/H₂ O/acetic acid ethyl ester(3:1:1:5), melting point 178° C. (decomposition).

[α]_(D) ²⁰ : -29.85° and [α]₅₄₆ ²⁰ : -36° (c=1, in DMF).

Yield: 3.72 g (96 percent of theoretical).

C₅₃ H₇₂ N₉ O₁₄ SBa₀.5.1H₂ O. 1 DMF (1235.10); Calculated: C: 54.46, H:6.61, N: 11.34, Found: C: 54.70, H: 6.73, N: 11.34.

C. B--Arg(B₂)--Asp(OtBu)--Tyr(SO₃Ba_(1/2))--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂.2H₂ O

0.36 g (3.16 mmole) N-hydroxysuccinimide and at -20° C. 0.63 g (3.04mmole) dicyclohexylcarbodiimide are added to 3 g (2.43 mmole) H--Tyr(SO₃Ba_(1/2))--Thr(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂.1H₂ O and 2.18 g(2.92 mmole) B--Arg(B₂)--Asp(OtBu)--OH in dimethylformamide. After 24hours at 4° C. and 24 hours at room temperature the reaction mixture isfiltered from the precipitated urea and concentrated in vacuum. Theresidue is reprecipitated twice from dimethylformamide-ether.

Chromatographically pure in n-BuOH/AcOH/H₂ O/acetic acid ethyl ether(3:1:1:5); melting point 190° to 195° C.

[α]_(D) ²⁰ : -14.3° and [α]₅₄₆ ²⁰ : -17.3° (c=1, in DMF).

D. H--Arg--Asp(OtBu)--Tyr(SO₃H)--Thr(tBu)--Gly--Trp--Leu--Asp--(OtBu)--Phe--NH₂

3.7 g (1.9 mmole) B--Arg(B₂)--Asp(OtBu)--Tyr(SO₃Ba_(1/2))--Thr--(tBu)--Gly--Trp--Leu--Asp(OtBu)--Phe--NH₂.2H₂ O were asusual hydrogenated in the presence of palladium in a solution ofdimethylformamide containing 26.34 ml 0.143 n HCl in MeOH (theor. 26.87ml) at pH 6.

After removal of the catalyst the product was precipitated with watercontaining triethylamine. The precipitate was reprecipitated fromdimethylformamide-water and carefully washed with water.

Chromatographically pure in n-BuOH/AcOH/H₂ O/acetic acid ethyl ester(3:1:1:5), melting point 208° C. (decomposition).

[α]₅₄₆ ²⁰ : -25.5° and [α]_(D) ²⁰ : -21.2° (c=1, in DMF).

Yield: 2.24 g (83 percent of theoretical).

C₆₇ H₉₇ N₁₄ O₁₈ S (1418.7): Calculated: C: 56.72, H: 6.89, N: 13.82,Found: C: 56.63, H: 6.92, N: 13.47.

EXAMPLE 4

In accordance with the method of the present invention the followingcompound was prepared:

    H--Arg--Asp--Tyr(SO.sub.3 H)--Thr--Gly--Trp--Nle--Asp--Phe--NH.sub.2 :

Aminoacid analysis (calculated values in brackets) of the acidhydrolysate (6 m HCl/110° C./24 hours with addition of 2.5 percentmercapto-acetic acid):

    Arg 1.00[1] Asp 1.92[2] Tyr 1.00[1] Thre 1.03[1] Gly 0.98[1] Trp 0.91[1] Nle 1.02[1] Phe 1.00[1];

of the AP-M disintegration:

    Arg 1.00[1] Asp 1.96[2] Tyr(SO.sub.3 H) 0.98[1] Thr 1.05[1] Gly 1.00[1] Trp 1.00[1] Nle 1.01[1] Phe 1.00[1];

thin layer chromatography (HPTLC-ready to use plates silicagel 60, MerckAG, Darmstadt, Germany) uniform in n-butanol/acetic acid/pyridine/water(60:6:40:24); uniform after high pressure liquid chromatography [column:μ-Bondapak C 18; eluent: 29% acetonitrile and 71% 0.01 molar ammoniumacetate buffer, pH 4.0; isocratic] and carrierless electrophoresis[Chamber electrolyte: 0.1 molar ammonium acetate buffer, pH 8.3;electrode distance 50 cm at 1500 volts]; Tyr/Trp=0.0 (according to theUV determination).

What is claimed is:
 1. In the method of preparing peptides containing atyrosine-O-sulfate moiety the improvement comprising the use oftyrosine-O-sulfate barium salt or a N-acyl derivative thereof in saidsynthesis.
 2. The improvement of claim 1 wherein said barium salt isN-carbobenzoxy-tyrosine-O-sulfate barium salt.
 3. The improvement ofclaim 1 wherein said barium salt isN-t-butyloxycarbonyl-tyrosine-O-sulfate barium salt.
 4. The improvementof claim 1 wherein said barium salt isN-fluorenyloxycarbonyl-tyrosine-O-sulfate barium salt.
 5. Theimprovement of claim 1 wherein said barium salt isN-2-nitrophenylsulfenyl-tyrosine-O-sulfate barium salt.